How much PhiX spike-in is recommended when sequencing low diversity libraries on Illumina platforms?


When sequencing libraries with low base diversity, unbalanced nucleotide composition can negatively impact cluster template registration on non-patterned flow cells, Q30 scores, and data output.

For more information on the importance of base diversity on Illumina sequencing platforms, refer to the bulletin What is nucleotide diversity and why is it important?

To compensate for low base diversity in libraries, Illumina recommends spiking in PhiX Control v3 Library (FC-110-3001, commonly referred to as “PhiX”) for sequencing. The PhiX Control v3 Library has a diverse base composition (45% GC and 55% AT) that provides the balanced fluorescent signals that low diversity sample libraries lack during each sequencing cycle. This, in turn, assists with template registration and improves overall run quality.

For more information about using PhiX Control v3 Library, refer to the bulletin What is the PhiX Control v3 Library and what is its function in Illumina Next Generation Sequencing?

This table lists the percentage of PhiX Control v3 library Illumina recommends spiking in when running low diversity libraries on the indicated sequencing platforms and control software versions.


Platform PhiX Aligned (%)†
iSeq™ 100 Minimum 5%
MiniSeq™ 10-50%*
MiSeq™ (MCS 2.2 or higher) Minimum 5%
NextSeq™ 500/550 10-50%*
NextSeq 1000/2000 10-50%*
HiSeq™ 1000/1500/2000/2500 (HCS 2.2.38 or higher) Minimum 5%
HiSeq 3000/4000 (HCS 3.3.76 or lower) 10-50%*
HiSeq 3000/4000 (HCS 3.4.0 or higher) 5-20%*
NovaSeq™ Minimum 5%


† Differences in clustering efficiency between PhiX and the sample library can affect the PhiX spike-in percentage required to achieve the above-targeted percent PhiX aligned. For example, more PhiX may be required if the sample library clusters more efficiently than PhiX. Email Technical Support at with any questions about your particular library and platform.

*PhiX can be further adjusted based on experimentation. Illumina recommends starting with higher spike-in percentages and reducing based on run performance.

Frequently Asked Questions:

  1. Can PhiX Control v3 Library provide high nucleotide diversity for low diversity indexes?
    No, PhiX Control v3 Library is unindexed and does not balance signals for index reads.
  2. Are there other considerations when sequencing low diversity libraries?
    Reduce the library loading concentration to target a cluster density 30–40% beneath the optimal range for the chemistry version and platform used. The optimal amount of reduction required must be empirically determined. For more information, refer to:
    Cluster density guidelines for Illumina sequencing platforms
    Optimizing Cluster Density on Illumina Sequencing Systems